Affiliation: Department of Genetics and Microbiology, University of Milan, 20133 Milan, Italy. degryse@scripps.edu

ABSTRACTHMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a G(i/o) protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage.

Figure 5: HMG1 binds to the surface of RSMC and stimulates cell motility through RAGE. (A) Large amounts of HMG1 bind to the surface of RSMC. 1 million cells were incubated at 4°C with 800 ng Box A+B and 5 μg BSA. P shows the proteins associated with the cells, after washing, and S shows 20 μl of the medium containing unbound protein. (B) RSMC express RAGE. 1 million cells were lysed on the plate in SDS-PAGE sample buffer, heated for 5 min at 100°C, and then loaded on a 12% tricine gel. RAGE was detected by Western blot with an anti–RAGE antibody. (C) Anti–RAGE antibody inhibits HMG1-induced RSMC migration. The value of 100% corresponds to the number of cells migrating in the absence of any stimulator (random cell migration). The data represent the mean ± SD (n = 3). Statistical significance was 0.001 < P < 0.0001 for treatment with HMG1 and HMG1 + unspecific antibody. Treatments with HMG1 + anti–RAGE antibody, and HMG1 + anti–HMG1 antibody, had no statistically significant difference from the control. Treatment with anti–HMG1 and anti–RAGE antibodies alone also did not differ from the control.

Mentions:
To act as a migratory signal, HMG1 must arrive to the membrane of responsive cells and bind to a receptor. To test whether HMG1 binds to the surface of RSMC, we incubated these cells at 4°C with the Box A+B polypeptide, which is slightly smaller than the endogenous, full-length HMG1 and can thus be distinguished easily on SDS-PAGE gels. From the amount of Box A+B recovered in the cell pellet and in the supernatant (Fig. 5 A), it can be estimated that as much as 7 million Box A+B molecules can bind to a single RSMC (see Materials and Methods). This result demonstrates that extracellular HMG1 (or truncated forms thereof) can bind to RSMC, but most likely does not reflect the actual receptor number. Indeed, HMG1 has already been shown to bind to heparin and proteoglycans (Bianchi 1988; Salmivirta et al. 1992; Nair and Jungalwala 1997); thus, HMG1 might also be associated with the extracellular matrix produced by RSMC. In accordance with this hypothesis, only small amounts of HMG1 bind to cells, like HeLa, that produce little extracellular matrix (not shown).

Figure 5: HMG1 binds to the surface of RSMC and stimulates cell motility through RAGE. (A) Large amounts of HMG1 bind to the surface of RSMC. 1 million cells were incubated at 4°C with 800 ng Box A+B and 5 μg BSA. P shows the proteins associated with the cells, after washing, and S shows 20 μl of the medium containing unbound protein. (B) RSMC express RAGE. 1 million cells were lysed on the plate in SDS-PAGE sample buffer, heated for 5 min at 100°C, and then loaded on a 12% tricine gel. RAGE was detected by Western blot with an anti–RAGE antibody. (C) Anti–RAGE antibody inhibits HMG1-induced RSMC migration. The value of 100% corresponds to the number of cells migrating in the absence of any stimulator (random cell migration). The data represent the mean ± SD (n = 3). Statistical significance was 0.001 < P < 0.0001 for treatment with HMG1 and HMG1 + unspecific antibody. Treatments with HMG1 + anti–RAGE antibody, and HMG1 + anti–HMG1 antibody, had no statistically significant difference from the control. Treatment with anti–HMG1 and anti–RAGE antibodies alone also did not differ from the control.

Mentions:
To act as a migratory signal, HMG1 must arrive to the membrane of responsive cells and bind to a receptor. To test whether HMG1 binds to the surface of RSMC, we incubated these cells at 4°C with the Box A+B polypeptide, which is slightly smaller than the endogenous, full-length HMG1 and can thus be distinguished easily on SDS-PAGE gels. From the amount of Box A+B recovered in the cell pellet and in the supernatant (Fig. 5 A), it can be estimated that as much as 7 million Box A+B molecules can bind to a single RSMC (see Materials and Methods). This result demonstrates that extracellular HMG1 (or truncated forms thereof) can bind to RSMC, but most likely does not reflect the actual receptor number. Indeed, HMG1 has already been shown to bind to heparin and proteoglycans (Bianchi 1988; Salmivirta et al. 1992; Nair and Jungalwala 1997); thus, HMG1 might also be associated with the extracellular matrix produced by RSMC. In accordance with this hypothesis, only small amounts of HMG1 bind to cells, like HeLa, that produce little extracellular matrix (not shown).

Bottom Line:
HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays.HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells.These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses.

Affiliation:
Department of Genetics and Microbiology, University of Milan, 20133 Milan, Italy. degryse@scripps.edu

ABSTRACTHMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a G(i/o) protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage.